Flea infestation of animals is a health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas directly cause a variety of diseases, including allergies, and also carry a variety of infectious agents including, but not limited to, endoparasites (e.g., nematodes, cestodes, trematodes and protozoa), bacteria and viruses. In particular, the bites of fleas are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects. As such, fleas are a problem not only when they are on an animal but also when they are in the general environment of the animal.
Bites from fleas are a particular problem because they not only can lead to disease transmission but also can cause a hypersensitive response in animals which is manifested as disease. For example, bites from fleas can cause an allergic disease called flea allergic (or allergy) dermatitis (FAD). A hypersensitive response in animals typically results in localized tissue inflammation and damage, causing substantial discomfort to the animal.
The medical importance of flea infestation has prompted the development of reagents capable of controlling flea infestation. Commonly encountered methods to control flea infestation are generally focused on use of insecticides. While some of these products are efficacious, most, at best, offer protection of a very limited duration. Furthermore, many of the methods are often not successful in reducing flea populations. In particular, insecticides have been used to prevent flea infestation of animals by adding such insecticides to shampoos, powders, collars, sprays, foggers and liquid bath treatments (i.e., dips). Reduction of flea infestation on the pet has been unsuccessful for one or more of the following reasons: (1) failure of owner compliance (frequent administration is required); (2) behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and (3) the emergence of flea populations resistant to the prescribed dose of pesticide. Flea populations, however, have been found to become resistant to insecticides.
20-Hydroxyecdysone (ecdysone) is the insect steroid hormone which regulates molting and metamorphosis. The ability of ecdysone to have a pleiotropic effect upon various tissues is dependent upon the formation of a complex of ecdysone with its receptor (EcR) and its heterodimeric partner, ultraspiracle (USP). This complex then binds to ecdysone response elements (EcRE) found within the promoters of insect genes, and thereby affecting DNA transcription. EcR by itself has been reported to be incapable of high affinity binding or transcriptional activation, rather, these activities appear to be dependent upon heterodimer formation with USP, Yao et al., 1993, Nature 366, 476-479.
Prior investigators have described certain insect EcR protein or nucleic acid sequences, including for example, Bombyx mori, Swevers et al., 1995, Insect Biochem. Mol. Biol. 25(7), 857-866; Drosophila melanogaster, Koelle et al., 1991, Cell 67(1), 59-77; and Manduca sexta, Fujiwara et al., 1995, Insect Biochem. Mol Biol. 25(7), 845-856; and certain insect USP protein and nucleic acid sequences, including for example, Bombyx mori, Tzertzinis et al., 1994, J. Mol. Biol. 238, 479-486; Drosophila melanogaster, Oro et al., 1990, Nature, 347(6290) 298-301; and Manduca sexta, Jindra et al., GenBank Accession 1718061 (SEQ ID NO:72). Prior investigators have also described mammalian homologs of EcR and USP, Giguere et al., 1987, Nature 330(6149), 624-629; Cooke et al., 1996, GenBank Accession 1350913 (SEQ ID NO:73); Leid et al., 1992, Cell 68(2), 377-395; and amphibian homologs, Blumberg et al., 1992, Proc. Natl. Acad. Sci., U.S.A. 89(6), 2321-2325.
Identification of flea EcR and USP of the present invention is surprising, however, due to the source from which these molecules were identified. Most lepidopterans and dipterans are better characterized, relative to C. felis, with respect to visible signs of molting, the only stages which should possess high levels of ecdysone. Ecdysone is necessary for the up regulation of mRNA encoding EcR and USP. Therefore, the lack of clear, easily visible signs of molting in C. felis make the likelihood of finding cDNA containing EcR or USP message in the larval and prepupal cDNA unexpected.
Thus, there remains a need to develop a reagent and a method to protect animals from flea infestation.